This invention pertains to a method and test kit for the detection of hepatitis B virus by nucleic acid hybridization. More specifically, the invention pertains to a method for detecting the presence of hepatitis B virus-DNA (hereinafter sometimes referred to as HBV-DNA) in the serum of a human or animal subject.
Still more specifically, the invention relates to a method for the direct detection of HBV-DNA in blood, blood products, vaccines, and other body fluids.
Over the years several tests have been employed to detect the presence of hepatitis B virus (hereinafter sometimes referred to as HBV) constituents in serum and other body fluids. These tests are primarily immunological in principle and depend on the presence of antibodies produced in humans or animals to detect specific viral proteins such as hepatitis B surface antigen HB.sub.s Ag), hepatitis B core antigen (HB.sub.c Ag) or hepatitis B "E" antigen (HB.sub.e Ag). Radioimmunoassay, considered to be the most sensitive immunological technique, employs 125 iodine-labelled antibody. Radioimmunoassay has sufficient sensitivity to detect nanogram (10.sup.-9 gram) quantities of HB.sub.s Ag. However, immunological tests are indirect, and nonspecific antigen-antibody reactions do occur resulting in false positive determinations. Furthermore, in certain instances the antigen-antibody tests are negative in donor serum, but the recipient of transfused blood develops hepatitis B virus infection. Despite careful radioimmunoassay of all blood donated in the U.S. for HB.sub.s Ag, a significant percentage of post-transfusion hepatitis cases are still caused by transfusion of blood that is contaminated with HBV which eluded detection. Hence, radioimmunoassay and other immunological tests have serious drawbacks, limited utility and provide only an indirect index of potential viral infectivity.
Among the other tests used to identify potentially infectious virus in serum are the viral polymerase assay and electron microscopy. For the most part, these methods are cumbersome assays of relatively low sensitivity and would be impractical for use as a routine laboratory screening procedure.
The present invention involves the discovery of a simple and sensitive mass screening procedure for hepatitis B virus in which molecular hybridization and recombinant DNA techniques are used to detect HBV-DNA directly in human serum. According to the invention, a hybridization probe is prepared by cloning hepatitis B virus-DNA. The cloned HBV-DNA is purified from the recombinant plasmid and labelled to high specific activity with .sup.32 P or .sup.125 I to form a hybridization probe. The hybridization probe is applied to a test sample (suspected of containing HBV) that has been fixed to a suitable substrate and the probe bearing sample incubated under hybridization conditions which permit the labelled HBV-DNA to hybridize (combine) with only HBV-DNA sequences present in the test sample. Following incubation, the uncombined HBV-DNA probe is removed from the substrate and the presence of hybridized HBV-DNA determined by liquid scintillation spectroscopy or by autoradiography of the substrate. A positive assay is evidence that the test sample contains the DNA of HBV.
This hybridization analysis is substantially more sensitive than methods presently utilized to detect HBV or HBV proteins in the serum or other body fluids. Moreover, the test provides direct evidence for viral infectivity by identifying the DNA of the viral particle, rather than by demonstrating the presence of viral protein antigens. The test is highly specific for HBV, virtually eliminates false positive results and can be applied to other DNA or RNA test systems for which a purified nucleic acid hybridization probe can be prepared. A principal advantage of the method of the present invention is that the sample to be tested can be used directly, without significant pretreatment. The components required to conduct the assay of the invention can be prepared in the form of a test kit, which can be utilized without difficulty by laboratory personnel with a minimum of training.
It is therefore an object of the present invention to provide a method for the detection of HBV in serum, or body fluids.
A further object of the present invention is to provide a sensitive mass screening method for the detection of HBV by demonstrating the presence of the unique DNA contained within this virus.
A still further object of the invention is to provide a method for identifying carriers of HBV who may elude detection by other tests.
A further object of the invention is to provide a simple test to distinguish individuals who have only viral proteins in their serum or body fluids from those patients who manifest potentially complete infectious virus as demonstrated by the presence of HBV-DNA.
Another object of the invention is to provide a method for assaying the level of infectious HBV-DNA in a patient's peripheral circulation or body fluids.
Another object of the present invention is to provide a test kit comprising an HBV-DNA hybridization probe, a detection substrate, and a sealable container for incubating the test reagents, and optionally the other components necessary to conduct the test; namely, incubation solutions.
References which pertain to the subject invention are:
Shouval et al., Clinical Research, Volume 28, No. 2, April 1980 (Abstract);
Chakraborty et al. Nature, Volume 286, No. 5772, pages 531-533, July 31, 1980.
Shouval et al., Proceedings National Academy of Science (PNAS) U.S.A., Volume 77, No. 10, pages 6147-6151, October 1980.
Bonino et al., Gastroenterology, Volume 79, No. 5, November 1980, page 1006.
Shafritz and Kew, Hepatology, Volume 1, No. 1, pages 1-8, Jan.-Feb. 1981.
U.S. Pat. No. 4,034,072.
U.S. Pat. No. 4,038,378.